The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2
The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2
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DOI：
10.1002/j.1460-2075.1996.tb01097.x
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发表时间：
1996-12-16
期刊：
影响因子：
11.4
通讯作者：
Taya, Y
中科院分区：
文献类型：
--
作者：
Kitagawa, M;Higashi, H;Taya, Y
细胞周期蛋白D-Cdk 4/6和细胞周期蛋白A/E-Cdk 2被认为在细胞周期的G（1）/S转换期间参与视网膜母细胞瘤蛋白（pRB）的磷酸化。然而，目前还不清楚为什么需要几个Cdk，以及它们之间的区别。我们发现细胞周期蛋白D1-Cdk 4磷酸化的共有氨基酸序列不同于S/T-P-X-K/R，S/T-P-X-K/R是细胞周期蛋白A/E-Cdk 2使用各种合成肽作为底物磷酸化的共有序列。Cyclin D1-Cdk 4能有效磷酸化含有pRB部分序列的G1肽RPPTLS（780）PIP-，而Cyclin E-Cdk 2和A-Cdk 2则不能。为了在体外和体内检测pRB的磷酸化状态，我们制备了pRB中磷酸化丝氨酸780的特异性抗体。我们证实，细胞周期蛋白D1-Cdk 4，而不是细胞周期蛋白E-Cdk 2，磷酸化丝氨酸780在重组pRB。pRB中的Ser 78 O在GI期以细胞周期依赖的方式被磷酸化，并且我们发现在Ser 780磷酸化的pRB在体内不能与E2 F-1结合。结果表明，cyclin D1-Cdk 4和cyclin A/E Cdk 2在体内磷酸化pRB的不同位点。
Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.